Means and methods for treating or preventing brain tumors based on the nuclear receptor tailless (tix)

ABSTRACT

The present invention relates to the use of an inhibitor of tailless gene expression or tailless protein activity for the preparation of a pharmaceutical composition for treating or preventing the brain tumor in a subject. Preferably an inhibitor of tailless expression is a single or double stranded RNA. An inhibitor of tailless protein activity is preferably an antibody which specifically binds thereto. Finally, the invention includes methods for identifying anti-brain tumor drugs.

This application is a divisional application of U.S. patent applicationSer. No. 13/774,359, filed Feb. 22, 2013, which is a divisionalapplication of U.S. patent application Ser. No. 12/744,802, filed Oct.25, 2010, now U.S. Pat. No. 8,404,657, which is a National Stage ofInternational Application No. PCT/EP20081065731, filed Nov. 18, 2008,which is based upon and claims benefit of priority from prior European.Patent Application No. 07121641.0, filed Nov. 27, 2007, the entirecontents of each of which are incorporated by reference herein in theirentireties.

The present invention is concerned with therapeutic measures for braintumors. Specifically, the present invention relates to the use of aninhibitor of tailless gene expression or tailless protein activity forthe preparation of a pharmaceutical composition for treating orpreventing the brain tumor in a subject. Preferably an inhibitor oftailless expression is a single or double stranded RNA. An inhibitor oftailless protein activity is preferably, an antibody which specificallybinds thereto. Finally, the invention includes methods for identifyinganti-brain tumor drugs.

The existence of a cancer stern cell population in human brain tumors ofdifferent phenotypes from both, children and adults, has been described(Vescovi 2006, Nat Rev Cancer 6: 425-436). However, the cellular originof brain tumors remains a subject of an ongoing debate in the scientificliterature. The decisive issue is whether brain tumors arise from thededifferentiation of a normal brain cell or from the transformation of anormal neural stem cell or progenitor cell.

The tailless (TIx) gene encodes an orphan nuclear receptor which isexpressed in the developing eye and brain and in adult neuralstem/progenitor cells. Tailless expressing cells also express CD 133, awidely used marker of brain tumor stem cells. Loss of tailless leads toa regulation of PTEN in the subventricular zone compatible with itsfunction in control of neural stem cell proliferation. A series ofstudies showed that tailless is significantly overexpressed in humanbrain tumors. The tumor types included neurocytoma, glioma andependymoma (Modena 2006, J Clin Oncol. 24: 5223-5233; Phillips 200$,Cancer Cell 9: 157-173; Sharma 2007, Cancer Research 67: 890-900; Sim2006, J Neuroscie 12544-12555; Taylor 2005, Cancer Cell 8: 323-335).

Efficient and reliable therapies and therapeutic approaches for thetreatment of brain tumors including neurocytoma, glioma and ependymomaas well as astrocytoma are not yet available but nevertheless highlydesirable.

The technical problem underlying the present invention is to providemeans and methods complying with those needs. The technical problem issolved by the embodiments characterized in the claims and herein below.

Accordingly, the present invention relates to the use of an inhibitor oftailless gene expression for the preparation of a pharmaceuticalcomposition for treating or preventing a brain tumor in a subject.

The term “inhibitor of tailless gene expression” relates to a compoundcapable of inhibiting the formation (i.e. transcription) of RNA encodingthe tailless nuclear receptor protein or to a compound which is capableof reducing the amount of such RNA present in a cell.

Preferably, the inhibitor abolishes the de novo formation or the amountof tailless encoding RNA to an extent that the amount of taillessnuclear receptor protein in a cell which has been brought into contactthe said inhibitor is significantly reduced compared to a cell which hasnot been contacted with the said inhibitor. Preferably, the compoundsuitable as an inhibitor of tailless gene expression is capable ofinteracting with a polynucleotide encoding the tailless nuclear receptorprotein.

Polynucleotides encoding the tailless protein as referred to inaccordance with the present invention are, preferably RNA molecules,more preferably mRNA molecules or precursors thereof. Also encompassedis, however, the genomic DNA of the tailless gene present in a cell. Theaforementioned polynucleotides are, preferably, human polynucleotides.More preferably, the polynucleotide comprises a nucleic acid sequence asshown in SEQ ID NO: 1 or is a polynucleotide comprising nucleic acidsequence encoding an amino acid sequence as shown in SEQ ID NO: 2. It isto be understood that the present invention also includes inhibitors ofvariants of the human tailless polynucleotides specifically referred tobefore. Variants are, preferably, polynucleotides comprising a nucleicacid sequence which is at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98% or at least 99%identical to the nucleic acid sequence shown in SEQ ID NO: 1 or nucleicacid sequences encoding an amino acid sequence as shown in SEQ ID NO: 2.It is to be understood that the variant polynucleotides must encode anuclear receptor having tailless function. Such polynucleotide variants,in particular, are represented by orthologs or paralogs of the taillessgene. The degree of sequence identity (in percentage values) as referredto above can be, preferably, determined by sequence comparisonalgorithms well known in the art and, in particular, by the algorithmsof Needleman and Wunsch or Smith and Waterman give particularly reliableresults. To carry out the sequence alignments, the program PileUp (J.MoI. Evolution., 25, 351-360, 1987, Higgins et al, CABIOS, 5 1989:151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. MoI.Biol. 48; -443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2;-482-489 (1981))], which are part of the CGC software packet [GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991)], areto be used. The sequence identity values recited above in percent (%)are to be determined, preferably, using the program GAP over the entiresequence region with the following settings: Gap Weight: 50, LengthWeight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which,unless otherwise specified, shall always be used as standard settingsfor sequence alignments.

Preferably, inhibitors of tailless gene expression are single or doublestranded RNA molecules which are capable of specifically binding to atailless polynucleotide referred to above. Such single or doublestranded RNA molecules are used for gene expression regulation by meansof antisense RNA or ribozyme technology or RNA interference (RNAi)technology). Other preferred inhibitors of tailess gene expression aretriple helix firming oligonucleotides, preferably DNA oligonucleotides.The molecular principles of the aforementioned technologies are wellknown in the art.

RNAi is mediated by small interfering RNAs (siRNAs). The term “smallinterfering RNA” or “siRNA” refers to a nucleic acid molecule which is adouble stranded RNA agent that is complementary to i.e., able tobase-pair with, a portion of a target RNA (generally mRNA), i.e. thepolynucleotide of the present invention being RNA. siRNA acts tospecifically guide enzymes in the host cell to cleave the target RNA. Byvirtue of the specificity of the siRNA sequence and its homology to theRNA target, siRNA is able to cause cleavage of the target RNA strand,thereby inactivating the target RNA molecule. Preferably, the siRNAwhich is sufficient to mediate RNAi comprises a nucleic acid sequencecomprising an inverted repeat fragment of the target gene and the codingregion of the gene of interest (or portion thereof). Also preferably, anucleic acid sequence encoding a siRNA comprising a sequencesufficiently complementary to a target gene is operatively linked to aexpression control sequence. Thus, the mediation of RNAi to inhibitexpression of the target gene can be modulated by said expressioncontrol sequence. Preferred expression control sequences are those whichcan be regulated by a exogenous stimulus, such as the tet operator whoseactivity can be regulated by tetracycline or heat inducible promoters.Alternatively, an expression control sequence may be used which allowstissue-specific expression of the siRNA. The complementary regions ofthe siRNA allow sufficient hybridization of the siRNA to the target RNAand thus mediate RNAi. In mammalian cells, siRNAs are approximately21-25 nucleotides in length. The siRNA sequence needs to be ofsufficient length to bring the siRNA and target RNA together throughcomplementary base-pairing interactions. The siRNA used with the Tetexpression system of the invention may be of varying lengths. The lengthof the siRNA is preferably greater than or equal to ten nucleotides andof sufficient length to stably interact with the target RNA;specifically 15-30 nucleotides; more specifically any integer between 15and 30 nucleotides, most preferably 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, and 30. By “sufficient length” is meant anoligonucleotide of greater than or equal to 15 nucleotides that is of alength great enough to provide the intended function under the expectedcondition. By “stably interact” is meant interaction of the smallinterfering RNA with target nucleic acid (e.g., by forming hydrogenbonds with complementary nucleotides in the target under physiologicalconditions). Generally, such complementarity is 100% between the siRNAand the RNA target, but can be less if desired, preferably 91%, 92%, 93%94%, 95%, 96% 97%, 98%, or 99%. For example, 19 bases out of 21 basesmay be base-paired. In some instances where selection between variousallelic variants is desired, 100% complementary to the target gene isrequired in order to effectively discern the target sequence from theother allelic sequence. When selecting between allelic targets, choiceof length is also an important factor because it is the other factorinvolved in the percent complementary and the ability to differentiatebetween allelic differences. Methods relating to the use of RNAi tosilence genes in organisms, including C. elegans, Drosophila, plants,and mammals, are known in the art (see, for example, Fire et al., Nature(1998) 391:806-811; Fire, Trends Genet. 1.5, 358-363 (1999); WO 0129058;WO 09932619).

The ribozyme- and antisense-based techniques for gene silencing are wellknown in the art (see Kalota 2004, Cancer Biology & Therapy 3:1 4-12).They are essentially based on antisense nucleic acid molecules orribozymes which comprise nucleic acid sequences which are complementaryto the target polynucleotides and, thereby, allow for specific bindingto the target polynucleotides. As a consequence of said specific bindingthe target polynucleotides will then be degraded or inactivated.Oligonucleotides as used herein, preferably, relate to small doublestranded DNA molecules which are either capable of binding to specificregions of a target genomic DNA whereby gene silencing is achieved(so-called triple helix forming oligonucleotides) or to oligonucleotideswhich act as decoys to sequester transcription factor specificallyrequired fur the transcription of a target gene. These techniques havealso been successfully used in vivo and also to some extend resultedalready in therapeutics (see also Kalota 2004 loc cit.).

More preferably the inhibitor of tailless expression is a single ordouble stranded RNA or an oligonucleotide which specifically binds to apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of:

-   -   a) a nucleic acid sequence as shown in SEQ ID NO: 1;    -   b) a nucleic acid sequence encoding an amino acid sequence as        shown in SEQ ID NO: 2; and    -   c) a nucleic acid sequence which is at least 70%, at least 80%,        at least 85%, at least 0%, at least 95%, at least 97%, at least        98% or at least 99% identical to the nucleic acid sequence of a)        or b) wherein that nucleic acid sequence encodes a tailless        polypeptide.

More preferably, the inhibitor of tailless expression is a single ordouble stranded RNA comprising or essentially consisting of a nucleicacid having a nucleotide sequence as shown in any one of SEQ ID NOs: 3to 6.

The term “pharmaceutical composition” as used herein comprises thecompounds of the present invention (i.e. inhibitors of tailless geneexpression/protein function) and optionally one or more pharmaceuticallyacceptable carrier. The compounds of the present invention can beformulated as pharmaceutically acceptable salts. Acceptable saltscomprise acetate, methylester, HCl, sulfate, chloride and the like. Thepharmaceutical compositions are, preferably, administered topically orsystemically. Suitable routes of administration conventionally used fordrug administration are oral, intravenous, or parenteral administrationas well as inhalation. However, depending on the nature and mode ofaction of a compound, the pharmaceutical compositions may beadministered by other routes as well. For example, nucleic acidcompounds may be administered in a gene therapy approach by using viralvectors or viruses or liposomes. Moreover, the compounds can beadministered in combination with other drugs either in a commonpharmaceutical composition or as separated pharmaceutical compositionswherein said separated pharmaceutical compositions may be provided inform of a kit of parts. The compounds are, preferably, administered inconventional dosage terms prepared by combining the drugs with standardpharmaceutical earners according to conventional procedures. Theseprocedures may involve mixing, granulating and compressing or dissolvingthe ingredients as appropriate to the desired preparation. It will beappreciated that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand the well-known variables. The carrier(s) must be acceptable in thesense of being compatible with the other ingredients of the formulationand being not deleterious to the recipient thereof. The pharmaceuticalcarrier employed may be, for example, either a solid, a gel or a liquid.Exemplary of solid carriers are lactose, terra alba, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and thelike. Exemplary of liquid carriers are phosphate buffered salinesolution, syrup, oil such as peanut oil and olive oil, water, emulsions,various types of wetting agents sterile solutions and the like.Similarly, the carrier or diluent may include time delay material wellknown to the art, such as glyceryl mono-stearate or glyceryl distearatealone or with a wax. Said suitable carriers comprise those mentionedabove and others well known in the art, see, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Thediluent(s) is/are selected so as not to affect the biological activityof the combination. Examples of such diluents are distilled water,physiological saline. Ringers solutions dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants, or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like. A therapeutically effectivedose refers to an amount of the compounds to be used in a pharmaceuticalcomposition of the present invention which prevents, ameliorates ortreats the symptoms accompanying a disease or condition referred to inthis specification. Therapeutic efficacy and toxicity of such compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., ED50 (the dose therapeutically effectivein 50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50. Thedosage regimen will be determined by the attending physician and otherclinical factors; preferably in accordance with any one of the abovedescribed methods. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. Progress can be monitored by periodicassessment. The pharmaceutical compositions and formulations referred toherein are administered at least once in order to treat or ameliorate orprevent brain tumors as recited in this specification. However, the saidpharmaceutical compositions may be administered more than one time, forexample from one to four times daily up to a non-limited number of days.Specific pharmaceutical compositions are prepared in a manner well knownin the pharmaceutical art and comprise at least one active compoundreferred to herein above in admixture or otherwise associated with apharmaceutically acceptable earlier or diluent. For making thosespecific pharmaceutical compositions, the active compound(s) willusually be mixed with a earlier or the diluent, or enclosed orencapsulated in a capsule, sachet, cachet, paper or other suitablecontainers or vehicles. The resulting formulations are to be adapted tothe mode of administration, i.e. in the forms of tablets, capsules,suppositories, solutions, suspensions or the like. Dosagerecommendations shall be indicated in the prescribers or usersinstructions in order to anticipate dose adjustments depending on theconsidered recipient.

The term “treating” means that the brain tumors or symptoms thereofreferred to in accordance with the present invention are at leastameliorated or, more preferably, even entirely removed in astatistically significant portion of subjects to which a compoundsuitable as an inhibitor of tailless gene expression or protein functionhas been administered. Whether a portion is statistically significantcan be determined without further ado by the person skilled in the artusing various well known statistic evaluation tools, e.g., determinationof confidence intervals, p-value determination, Student's t-test,Mann-Whitney test, etc.. Preferred confidence intervals are at least90%, at least 95%, at least 97%, at least 98% or at least 99%. Thep-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.

The term “preventing” as used herein means that the de novo formation ofbrain tumors can be abolished in a statistically significant portion ofsubjects to which the compounds suitable as a inhibitor of tailless geneexpression or protein function have been administered.

The term “brain tumor” relates to a tumor which arises from tissues ofthe nervous system and, preferably, of the central system. Morepreferably, a brain tumor in accordance with the present invention is aglioma, neurocytoma, astrocytoma or ependymoma. Most preferably, thetumor arises from CD 133 positive cancer stem cells.

The term “subject” as used herein preferably relates to mammals and mostpreferably to humans.

It has been found in accordance with the studies underlying the presentinvention that the orphan nuclear receptor tailless has oncogenicpotential rather than being merely a marker for brain tumors. Moreover,it was found that inhibiting tailless function in adult neural stemcells or brain tumor cells by interfering with the tailless geneexpression or the tailless nuclear receptor protein function will leadto treatment or prevention of brain tumors including glioma,neurocytoma, astrocytoma or ependymoma. Specifically, transgenic miceexpressing two copies of the tailless gene were generated in accordancewith studies underlying this invention. These mice developed braintumors from adult neuronal stem cells. The observed brain tumorsincluded glioma-like structures and other tumors referred to inaccordance with the present invention. As discussed above already,tailless was also found to be upregulated in gene expression profiles ofvarious brain tumors (Modena 2006, loc cit; Phillips 2006, loc cit.,Sharma 2007, loc cit, Sim 2006, loc cit., Taylor 2005 loc cit.).Specifically, a recent study showed that 50% of the analyzedglioblastomas exhibited tailless overexpression on the mRNA level.Contrary, mice lacking tailless in adult neuronal stem cells due to aconditional knock out of the tailless gene in the adult brain alsoshowed significantly reduced neurogenesis. These data show thattailless, indeed, is a molecular regulator of neurogenesis in the adultbrain including impaired neurogenesis which occurs in brain tumorformation. Moreover, thanks to the means and methods provided inaccordance with the present invention, tailless can be addressed as amolecular target for brain cancer therapies. Moreover it has been foundthat the therapeutic means and measures referred to herein will be alsoeffective against brain tumors which are derived from CD 133 positivecancer stem cells. Such tumors are normally not affected by conventionaltumor therapies such as chemotherapy or irradiation.

From the above it is to be understood that the present invention alsocontemplates the use of an inhibitor of tailless protein activity forthe preparation of a pharmaceutical composition for treating orpreventing a brain tumor.

The term “inhibitor of tailless protein activity” encompasses compoundswhich interfere with the tailless protein activity. Preferably, suchcompounds specifically bind to the tailless nuclear receptor proteinthereby inhibiting its capability to bind to its natural bindingpartners. In this context, the tailless protein is known to form homo-or heterodimers with of the nuclear receptor proteins to exert itsbiological functions. Such homo- or heterodimerization can be preventedby antibodies specifically binding to a tailless nuclear receptorprotein or by dominant negative nuclear receptor proteins known tointeract with tailless protein including tailless itself (see Zhu 1996,Endocrinology 137(2): 712-721).

More preferably, the inhibitor is an antibody which specifically bindsto a protein encoded by a polynucleotide having a nucleic acid sequenceselected from the group consisting of:

-   -   a) a nucleic acid sequence as shown in SEQ ID NO: 1;    -   b) a nucleic acid sequence encoding a polypeptide haying an        amino acid sequence as shown in SEQ ID NO: 2; and    -   c) a nucleic acid sequence being at least 70% identical to the        nucleic acid sequence of a) or b) wherein said sequence encodes        a tailless protein.

Antibodies against the tailless protein according to the invention canbe prepared by well known methods using a purified tailless protein or asuitable fragment derived therefrom as an antigen. A fragment which issuitable as an antigen may be identified by antigenicity determiningalgorithms well known in the art. Such fragments may be obtained eitherfrom the polypeptide of the invention by proteolytic digestion or may bea synthetic peptide. Preferably, the term “antibody” as used inaccordance with the present invention is a monoclonal antibody, ispolyclonal antibody, a single chain antibody, a human or humanizedantibody or primatized, chimerized or fragment thereof. Also comprisedby the term “antibodies” are a bispecific antibody, a syntheticantibody, an antibody fragment, such as Fab, Fv or scFv fragments etc.,or a chemically modified derivative of any of these. Antibodies orfragments thereof can be obtained by using methods which are described,e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press,Cold Spring Harbor, 1988, Monoclonal antibodies can be prepared by thetechniques originally described in Kóhler and Milstein, Nature 256(1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise thefusion of mouse myeloma cells to spleen cells derived from immunizedmammals. The said antibody shall specifically bind (i.e. does not crossreact with other polypeptides or peptides) to the tailless protein.Specific binding can be tested by various well known techniques.Moreover, the antibodies useful as compounds capable of inhibitingtailless protein function can be tested for this property in functionalassays such as nuclear localization assays. An antibody shall be capableof inhibiting tailless protein function if it prevents nuclearlocalization. Moreover, dimerization can be analyzed. An antibody shallbe capable of inhibiting tailless protein function if it prevents homo-or heterodimerization of the tailless protein.

The present invention finally also contemplates a method for treating orpreventing brain tumors in a subject comprising administering to asubject suffering from a brain tumor an inhibitor of tailless geneexpression or an inhibitor of tailless protein activity in atherapeutically effective amount. Preferred embodiments of the saidmethod have been discussed in connection with the uses of the presentinvention above.

Moreover, the present invention contemplates a method for identifying ananti-brain tumor drug comprising:

-   -   a) contacting a host cell expressing the tailless gene with a        compound suspected to be a candidate anti-brain tumor drug;    -   b) determining the amount of tailless protein in said host cell;        and    -   c) comparing the amount of tailless protein found in the said        host cell to the amount of tailless found in a reference host        cell which has not been brought into contact with the said        compound suspected to be a candidate anti-tumor drug wherein a        decreased amount of tailless protein found in the host cell        compared to the reference host cell is indicative for a compound        being a candidate anti-brain tumor drug.

Further, the present invention includes a method for identifying ananti-brain tumor drag comprising:

-   -   a) contacting a host cell comprising the tailless protein with a        compound suspected to be a candidate anti-brain tumor drug;    -   b) determining the biological activity of the tailless protein        in said host cell; and    -   c) comparing the biological activity of tailless protein found        in the said host cell to the biological activity of the tailless        protein found in a reference host cell which has not been        brought into contact with the said compound suspected to be a        candidate anti-tumor drug, wherein a decreased activity found in        the host cell compared to the reference host cell is indicative        for a compound being a candidate anti-brain tumor drug.

The expression “a compound suspected to be a candidate anti-brain tumordrug” are in accordance with the aforementioned method, preferably,compounds including small molecule compounds which are capable ofspecifically interfering with the transcription or translation of thetailless gene. Preferred compounds are the single or double stranded RNAmolecules or oligonucleotides referred to elsewhere in thisspecification. Further compounds are those which interfere with thebiological activity of the tailless protein. Preferred compounds in sucha context are small molecules or the antibodies and tailless mutantproteins referred to elsewhere in this specification. Depending on thechemical nature of the compounds the said compounds may be diluted inthe medium comprising the host cells or may be delivered by knownvehicles such as liposomes and the like. By “contacting” it is meantthat the host cell is brought into physical contact for a time periodsufficient for allowing interaction of the tailless polynucleotidetranscripts or the tailless polypeptides present in the said host cell.

The term “host cell” as used herein refers to a host cell which iscapable of expressing the tailless gene, i.e. a host cell which allowsfor transcription of the tailless gene into a tailless encoding mRNA andthe translation of the said mRNA into the tailless protein. Preferably,the term relates to a eukaryotic host cell, more preferably to amammalian host cell and, most preferably, to a human host cell. It is tobe understood that tailless gene expression includes either naturallyoccurring tailless gene expression which can be found in brain cellsand, in particular, in adult neuronal stem cells or brain tumor cellsincluding those specifically mentioned herein or heterologous expressionof the tailless gene, e.g., transient expression from an expressionvector comprising the tailless gene or cDNA or expression from atransgene which has been stably integrated into the host cell genome.

The amount of tailless protein can be determined by various techniquesfor protein detection known in the art. These techniques include thedetection of the amount of the tailless protein by antibodiesspecifically recognizing the tailless protein or chromatographicmethods. Also encompassed are, however, methods which aim to determinethe amount of tailless protein by measuring specific physico-chemicalparameters of the said protein, such as NMR- or mass spectrometry(MS)-based techniques. The amount of tailless protein can, of course,also be determined indirectly, i.e. by measuring its biologicalactivity. The amount, thus, determined will be compared to the amount oftailless protein determined by the same technique in a reference hostcell. The comparison may be carried out manually or computer-assisted. Adecrease in the amount or activity in the treated host cell (i.e. thehost cell which has been contacted to the putative drug) will beindicative for a compound which is capable of interfering with taillessgene expression or the tailless biological activity and, thus, acompound suitable as a candidate anti-brain tumor drug.

All references referred to above are herewith incorporated by referencewith respect to their entire disclosure content as well as theirspecific disclosure content explicitly referral to in the abovedescription.

The figures show:

FIG. 1: Neurogenesis in the adult SVZ is TIx dosage dependent; Mice withdifferent copy number of the TIx gene show that neurogenesis in theadult SVZ is TIx dosage-dependent.

FIG. 2: Overexpression of TIx leads to Brain tumor formation; (A)Development of brain tumors in the SVZ of TIx overexpressing mice; (B)Tumor cells are migrating along white matter tracts (arrow); (C) Tumorcells surround neurons.

The invention will now be illustrated by the following examples whichare not intended to restrict or limit the scope of this invention.

EXAMPLE 1 Analysis of TIx Overexpressing Mice

To mimic overexpression of TIx in human brain tumors, a transgenic mouseoverexpressing TIx was generated. A bacterial artificial chromosome(BAC)-based technology was applied, which means that the overexpressionof TIx is derived by the endogenous TIx regulatory sequences. These ledto specific overexpression of TIx in neural stem cells in which theendogenous gene is expressed. TIx-overexpressing mice with increasingcopy number of the transgene show that neurogenesis in the adult is TIxdosage-dependent (FIG. 1). The increased neurogenesis in TIxoverexpressing mice indicates that TIx can promote self-renewal of adultneural stem cell.

Interestingly the 2 copy TIx transgenic line starts to develop braintumors when they are about 1 year old (FIG. 2A) with characteristics ofhuman brain tumors. The tumor cells are migrating along the white mattertracts (FIG. 2 B) and they surround neurons (FIG. 2 C). The mice alsodevelop glioma-like lesions. These glioma-like structures express GFAPand DCX which are characteristics of human glioma cells. The celldensity in these structures is much higher than in normal brain tissueand they express the cell proliferation marker Ki67, which indicatesthat these cells are highly proliferating.

Since TIx was overexpressed only in neural stem cells, brain tumorformation in the TIx overexpressing line indicates that this brain tumoris a stem cell disease. This is the first mouse model which directlyproves that a brain tumor arises from a mutated stem cell population.

To determine whether self-renewal of adult neural stem cells depends onTIx expression, an inducible TIx knock-out mouse was analyzed bycrossing the TIx-CreERT2 mice with the mice in which the TIx gene isfloxed. Two weeks after induction of recombination in the adult mousebrain, neurogenesis in the SVZ is dramatically decreased as indicated byBrdU pulse labeling and DCX staining (FIGS. 2 A, B). Four weeks afterinduction there is basically no cell division in the SVZ suggesting thatadult neurogenesis in the SVZ depends on TIx expression (FIG. 2 C).Taken together with the TIx expression pattern, it was concluded thatexpression of TIx is required for the self-renewal of adult neural stemcells.

In summary, it was shown that the nuclear receptor Tailless is playing acentral and decisive role in regulating self-renewal of adult neuralstem cells. TIx overexpression stimulates brain tumor formation fromadult neural stem cells. These findings allow for a direct targeting ofbrain tumor stem cells by interfering with TIx function. It is alsopossible to detect the initiating stages of brain tumor formation byanalyzing TIx expression.

EXAMPLE 2 Inhibitors of Tailless Gene Expression

The following Oligonucleotides can be used to knock down the taillessgene expression:

SEQ ID No: start Sequence (DNA) Region GC % 3 389GGCTGAAGAAGTGTTTGGAAGTCAA ORF 44.0 4 833 GGAGAGAACTGTTTGTTCTAGGAAT ORF40.0 5 870 CATTCCGGTTGATGCTAACACTCTA ORF 44.0 6 926CAGATTCCCAGAAGCTGAACAAGAT ORF 44.0

“Start” indicates the nucleotide position with respect to the cDNAsequence of human tailless. The oligonucleotides will be chemicallysynthesized as double stranded oligonucleotides or siRNA. Theoligonuoleotides or siRNA molecules will be formulated in apharmaceutical acceptable manner and can be introduced into brain tumortissue as described in, e.g., Kalota loc. cit.. Upon uptake of theoligonucleotides or siRNA molecules, the brain tumor cells will stopexpressing tailless. As a consequence thereof, the brain tumor willretard as can be monitored by clinical diagnostic techniques such asPET.

1. A method for identifying an anti-brain tumor drug comprising; a)contacting a host cell expressing the tailless gene with a compoundsuspected to be a candidate anti-brain tumor drug; b) determining theamount of tailless protein in said host cell; and c) comparing theamount of tailless protein found in the said host cell to the amount oftailless found in a reference host cell which has not been brought intocontact with the said compound suspected to be a candidate anti-tumordrug wherein a decreased amount of tailless protein found in the hostcell compared to the reference host cell is indicative for a compoundbeing a candidate anti-brain tumor drug.
 2. A method for identifying ananti-brain tumor drug comprising; a contacting a host cell comprisingthe tailless protein with a compound suspected to be a candidateanti-brain tumor drug; b) determining the biological activity of thetailless protein in said host cell; and c) comparing the biologicalactivity of tailless protein found in the said host cell to thebiological activity of the tailless protein found in a reference hostcell which has not been brought into contact with the said compoundsuspected to be a candidate anti-tumor drug, wherein a decreasedactivity found in the host cell compared to the reference host cell isindicative for a compound being a candidate anti-brain tumor drug.